In studies described in the previous annual report, we showed that ulcerative colitis is associated with the presence of "non-classical" NKT cells that produce IL-13. The latter can then act as an autocrine factor that induces the NKT cells to manifest greatly increased cytotoxicity for epithelial cells. In addition, in collaborative work with investigators in Germany, we have also shown that the IL-13 induces decreases in the barrier function of the epithelium, thereby exposing the lamina propria to commensal organisms and antigens that induce inflammatory responses. Thus, it is very probable that these IL-13 effects are key factors in the causation of ulcers in ulcerative colitis. In the present period we conducted studies to investigate the mechanisms of IL-13 signaling wiht the view of understanding how such signaling affects NKT cell and epithelial cell function. In addition, we wished to explore how IL-12 induces TGF-beta and TGF-beta-mediated fibrosis, which are also accompaniments of ulcerative colitis. In initial studies we showed that activation of a TGF-beta-luciferase reporter gene in THP-1 (macrophage) cells could be obtained by stimulation with IL-13 and TNF-alpha, but not with either cytokine alone. In addition, we showed that induction of IL-13Ralpha2 required both cytokines signaling via STAT6 and NF-kappaB; however, once IL-13Ralpha2 expression is achieved, IL-13 can activate the TGF-beta1 reporter gene alone (in the absence of TNF-alpha) in a STAT6 independent fashion. For this process, the full length IL-13Ralpha2 molecule is necessary, as the deletion of the cytoplasmic tail abolishes TGF-beta1 reporter gene activation. Applying the above data to in vivo studies of IL-13 signaling, we found that blockade of TNF-alpha signaling by administration of TNF-alphaR-Fc (etanercept) in oxazalone-colitis and in bleomycin-induced lung fibrosis led to marked downregulation of IL-13Ralpha2 expression, TGF-beta1 production and collagen deposition. In addition, blockade of TNF-alpha signaling in oxazalone colitis, a mouse model of ulcerative colitis (again with etanercept) led to loss of IL-13Ralpha2 expression and vastly decreased TGF-beta1 production. In addition, it led to extension of the colitis to the proximal colon where it normally does not occur because of high TGF-beta1 production in this area. These studies indicate that stimulation of TGF-beta1 (and induction of fibrosis) by IL-13 and TNF-alpha is a two-stage process involving: 1) induction of the IL-13Ralpha2 and 2) induction of TGF-beta1 via IL-13Ralpha2 signaling. Thus, the IL-13Ralpha2 proves to be a signaling receptor (not just a decoy) that enables facilitated IL-13 signaling leading to TGF-beta1 production and fibrosis. As such it paradoxically serves as a therapeutic target for the prevention of tissue fibrosis during chronic inflammation. In addition, they show that a model of ulcerative colitis can be influenced by an agent (etanercept) that modulates the expression of the IL-13Ralpha2.